Conclusion
This work establishes a method to generate human iTregs which maintain stable phenotype and function upon in vitro re-stimulation. Further validation in pre-clinical models will be needed to ensure its suitability for applications in adoptive transfer.
Methods
We assess iTregs generated with various concentrations of rapamycin and differing ratios of α-CD3/CD28 beads for their differentiation, stability, expression of Treg signature molecules and T helper effector cytokines, and Treg-specific demethylation region (TSDR) status.
Results
iTregs generated in the presence of TGF-β, ATRA, rapamycin and a higher ratio of α-CD3/CD28 beads were highly suppressive and stable upon in vitro re-stimulation. These iTregs exhibited a similar expression profile of Treg signature molecules and T helper effector cytokines to nTregs, in the absence of TSDR demethylation.